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BOR - Papers in Press, published online ahead of print October 24, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.063446
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BIOLOGY OF REPRODUCTION 78, 243–253 (2008)
DOI: 10.1095/biolreprod.107.063446
© 2008 by the Society for the Study of Reproduction, Inc.

Ovary; 

Growth Differentiation Factor 9 (GDF9) Stimulates Proliferation and Inhibits Steroidogenesis by Bovine Theca Cells: Influence of Follicle Size on Responses to GDF91

Leon J Spicer 2 3, Pauline Y Aad 3, Dustin T Allen 3, Sabine Mazerbourg 4, Anita H Payne 4, and Aaron J Hsueh 4

Department of Animal Science,3 Oklahoma State University, Stillwater, Oklahoma 74078 Department of Obstetrics and Gynecology,4 Stanford University School of Medicine, Stanford, California 94305

ABSTRACT

Ovarian follicular development is controlled by numerous paracrine and endocrine regulators, including oocyte-derived growth differentiation factor 9 (GDF9), and a localized increase in bioavailable insulin-like growth factor 1 (IGF1). The effects of GDF9 on function of theca cells collected from small (3–6 mm) and large (8–22 mm) ovarian follicles were investigated. In small-follicle theca cells cultured in the presence of both LH and IGF1, GDF9 increased cell numbers and DNA synthesis, as measured by a 3H-thymidine incorporation assay, and dose-dependently decreased both progesterone and androstenedione production. Theca cells from large follicles had little or no response to GDF9 in terms of cell proliferation or steroid production induced by IGF1. Small-follicle theca cell studies indicated that GDF9 decreased the abundance of LHR and CYP11A1 mRNA in theca cells, but had no effect on IGF1R, STAR, or CYP17A1 mRNA abundance or the percentage of cells staining for CYP17A1 proteins. GDF9 activated similar to mothers against decapentaplegics (SMAD) 2/3-induced CAGA promoter activity in transfected theca cells. Small-follicle theca cells had more ALK5 mRNA than large-follicle theca cells. Small-follicle granulosa cells appeared to have greater GDF9 mRNA abundance than large-follicle granulosa cells, but theca cells had no detectable GDF9 mRNA. We conclude that theca cells from small follicles are more responsive to GDF9 than those from large follicles and that GDF9 mRNA may be produced by granulosa cells in cattle. Because GDF9 increased theca cell proliferation and decreased theca cell steroidogenesis, oocyte- and granulosa cell-derived GDF9 may simultaneously promote theca cell proliferation and prevent premature differentiation of the theca interna during early follicle development.

follicle, follicular development, gene expression, growth factors, growth differentiation factor 9 (GDF9), insulin-like growth factor 1 (IGF1), LH, ovary, steroidogenesis, theca cells

INTRODUCTION

Intraovarian factors derived from the theca layer, granulosa cells, and oocytes play important roles in stimulating and/or inhibiting theca cell functions during ovarian follicular development [1–4]. Among these intraovarian factors is growth differentiation factor 9 (GDF9), a member of the transforming growth factor beta (TGFB) superfamily that also includes various bone morphogenetic proteins (BMP) and activins [4–6]. Although GDF9 mRNA is primarily localized in oocytes of primary to larger follicles in cattle [7, 8], sheep [9], pigs [10], rodents [11–13], and primates [14, 15], GDF9 may be produced by granulosa cells in some species [10]. Proteins in the TGFB family initiate signaling by assembling type I and type II serine/threonine kinase receptor complexes that activate similar to mothers against decapentaplegics (SMAD) transcription factors [6, 16, 17]. In particular, GDF9 activates SMAD2/3 transcription factors in granulosa cells by activating the BMP type II receptor (BMPR2) and the type I receptor activin receptor-like kinase 5 (ALK5) [18–20]. However, the changes in theca cell ALK5 mRNA during follicular growth and the precise intracellular signaling system used by theca cells have not been evaluated.

Studies on mutant mice [12, 21] and sheep [22] with GDF9 gene defects, together with in vitro and in vivo studies [9, 23–29], indicate an important role of GDF9 in the stimulation of early follicular growth, cumulus expansion, and fertility. Regarding theca cells, ovaries from GDF9-null mice show a developmental block at the primary follicle stage characterized by a failure to form the theca layer in early follicles [23]. Moreover, treatment with GDF9 inhibits progesterone production by cultured rat [30] and human [31] theca cells. Although GDF9 treatment decreases forskolin-induced androgen production by human theca cells [31], GDF9 treatment increases androgen production in rat theca cells [30], indicating possible species differences in theca cell responses to GDF9. However, the role of GDF9 in regulating theca cell proliferation has not been studied, nor have the effects of GDF9 on the function of theca cells from different stages of follicle development been compared previously. Using cultures of pure thecal cells isolated from bovine follicles at different developmental stages, the present study investigated the effect of GDF9 treatment on insulin-like growth factor 1 (IGF1)-induced cell proliferation and steroidogenesis in cultured bovine theca cells and intracellular pathways activated by GDF9. Developmental differences in ALK5 mRNA in theca cells were also evaluated. In addition, to ascertain the intrafollicular source of GDF9 in bovine follicles, GDF9 mRNA was measured in granulosa and theca cells.

MATERIALS AND METHODS

Cell Culture

Ovaries from nonpregnant beef cows were collected from a local slaughterhouse, and based on surface diameter, theca cells were collected from small (3–6 mm) and large (8–22 mm) follicles as previously described [32, 33]. This size classification was based on previous observations indicating that 1) follicles ≥8 mm in diameter have much greater androstenedione and estradiol concentrations than small follicles [34, 35], 2) follicles that are destined to ovulate average 10 ± 2 mm surface diameter [36], and 3) similar classifications have been used previously to inventory follicles during bovine estrous cycles [37, 38]. Small and large follicles were bisected after aspiration of follicular fluid, their diameter was confirmed, and granulosa cells were separated from the theca interna via blunt dissection. The theca interna was torn into small pieces, rinsed with basal medium, and enzymatically digested for 1 h at 37°C on a rocking platform as previously described [32, 33, 39]. The nondigested tissue was filtered out through sterile syringe filter holders with metal screens of 149 µm mesh (Gelman, Ann Arbor, MI). Theca cells were then centrifuged at 50 x g for 5 min, the supernatant was discarded, and the pellet was washed with serum-free medium. The purity of theca cells prepared this way was >90% [40, 41] and was verified in the present study (see below). The theca cells were resuspended in serum-free medium containing collagenase or DNase (Sigma Chemical Co., St. Louis, MO) at 1.25 mg/ml or 0.5 mg/ml, respectively, to prevent cell clumping less than 1 h before plating.

Approximately 2.0 x 105 viable cells were plated on 24-well Falcon multiwell plates (Becton Dickinson, Lincoln Park, NJ) in 1.0 ml of basal medium (1:1 Dulbecco modified Eagle medium and Ham F-12 medium) containing 10% fetal calf serum (FCS), gentamycin (0.12 mM), glutamine (2.0 mM), and sodium bicarbonate (38.5 mM; all obtained from Sigma Chemical Co.). Just prior to plating, viability of theca cells from small and large follicles was determined by trypan blue exclusion and averaged 99% ± 2% and 91% ± 4%, respectively. Cells were cultured in an atmosphere of 95% air and 5% CO2 at 38.5°C in 10% FCS for the first 48 h, with a medium change at 24 h. Cells were then washed twice with serum-free medium (0.5 ml) and the various treatments (see below) were applied in serum-free medium (1.0 ml) for 48 h, with a medium change at 24 h, unless stated otherwise. After the second 24-h treatment interval, medium was collected for steroid radioimmunoassay (RIA), and cells were collected for enumeration (see below).

Reagents

The hormones used for cell culture were LH (bovine L1914; LH activity 2.0 x NIH-LH-S1 U/mg) from Scripps Laboratories (San Diego, CA), recombinant human IGF1 and BMP4 from R&D Systems (Minneapolis, MN), and recombinant rat GDF9 generated and characterized as previously described [24–26]. Briefly, expression vectors for wild-type and epitope-tagged GDF9 were constructed using pcDNA3.1 Zeo (Invitrogen Corp., Carlsbad, CA). N-tagged GDF9 encoded a Flag epitope (DYKDDDDK) for the M1 antibody followed by six histidine residues fused to the amino terminus of mature GDF9. Clonal human embryonic kidney 293T cell lines stably expressing wild-type and tagged GDF9 were used. Quantification of N-tagged GDF9 was conducted after purification with nickel column and measurement of protein content using a Micro BCA protein assay kit (Perstorp Life Science, Rockford, IL). Purified N-tagged GDF9 was then used as a standard for the quantification of wild-type GDF9 in the conditioned medium of 293T cells by immunoblots using specific GDF9 antibodies.

Experimental Design

To evaluate the dose-response effect of GDF9 on steroidogenesis and/or proliferation of small- and large-follicle theca cells, cells from small and large follicles were cultured for 48 h in 10% FCS, washed twice with serum-free medium as described earlier, and 0, 150, 300, and 600 ng/ml of GDF9 were applied for 48 h in the presence of either LH (30 ng/ml) or LH plus IGF1 (30 ng/ml). An additional experiment evaluated 2-day treatments with LH (30 ng/ml), IGF1 (30 ng/ml), or both on steroidogenesis and cell proliferation of large-follicle theca cells. The doses of hormones were selected based on previous studies indicating these doses are maximal for stimulation of steroidogenesis and cell proliferation [25, 32, 33].

To evaluate the effect of GDF9 on DNA synthesis of theca cells, cells from small follicles were cultured for 48 h in 10% FCS, serum-starved for 24 h in serum-free medium, and then cultured for 40 h in the presence of 37 000 Bq of 3H-thymidine with LH (0 or 30 ng/ml), IGF1 (0 or 30 ng/ml), and/or GDF9 (0 or 600 ng/ml) as previously described [20, 42], with the following modifications. Briefly, after 40 h, medium was aspirated, cells were washed twice with 0.5 ml of 0.9% saline and solubilized with 0.5 ml of 1 N NaOH (10 min at 25°C), and cell-associated [methyl-3H]-thymidine (GE Healthcare, Buckinghamshire, UK) was quantified by beta counting (Tri-Carb, Liquid Scintillation Analyzer, Model 1900CA; Packard, Downers Grove, IL).

To evaluate the effect of GDF9 on the percentage of cells staining for 17 alpha-hydroxylase-1 (CYP17A1) protein, small-follicle theca cells were cultured on 8-chamber slides (8 mm x 10 mm; Lab-Tek #177402; Nalge Nuc International, Naperville, IL) as described for 24-well culture plates, except that 1 x 105 cells were plated and 400 µl of medium used, rather than 1 ml, with the following treatments applied for 48 h in serum-free medium (containing 30 ng/ml of IGF1 and LH) after the initial 48-h plating in 10% FCS: control (no additions) or GDF9 (300 ng/ml). Medium was changed after 24 h. At the end of the 48-h treatment period, cells/slides were processed as described below.

To evaluate the purity of the theca cell preparations, levels of CYP17A1, aromatase (CYP19A1), and FSH receptor (FSHR) mRNA were measured in freshly isolated theca and granulosa cells from small and large follicles from six separate pools of cells. Procedures for RNA isolation and target gene mRNA abundance quantification are described below.

To compare the effects of GDF9 and BMP4 on activity of the SMAD binding element, CAGA, and the BMP response element (BRE) in small-follicle theca cells, cells were cultured as described earlier, with the following treatments applied for 24 h in serum-free medium (containing 30 ng/ml of IGF1 and LH) after a 4-h transfection (see below) with either CAGA or BRE promoters: control (no additions), GDF9 (300 ng/ml), or BMP4 (200 ng/ml). The doses of hormones were selected based on the results of previous in vitro studies [20, 25, 26, 43, 44] indicating these doses are effective in altering steroidogenesis and/or promoter activity in granulosa cells.

To test the ability of GDF9 to regulate the LH receptor (LHR) mRNA, IGF1 receptor (IGF1R) mRNA, steroidogenic acute regulatory protein (STAR) mRNA and the steroidogenic enzymes, side-chain cleavage enzyme (CYP11A1), and CYP17A1 mRNAs in cultured bovine theca cells, theca cells were obtained from small bovine follicles and cultured for 48 h in 10% FCS, followed by various hormonal treatments in serum-free medium as described above. Treatments applied for 12 h in serum-free medium (containing 30 ng/ml of IGF1 and LH) were: Control (no other additions), GDF9 (300 ng/ml), or BMP4 (200 ng/ml). At the end of treatment, cellular RNA was isolated as described below.

To determine the effects of LH alone, IGF1 alone, or LH plus IGF1 on abundance of LHR, CYP11A1, and IGF1R mRNA in theca cells, cells from large follicles were cultured for 48 h in 10% FCS, followed by 48-h treatment of LH (30 ng/ml), IGF1 (30 ng/ml), or both in serum-free medium. Medium was changed every 24 h, and cellular RNA was isolated at 48 h as described below.

To determine if ALK5 mRNA abundance differed between small- and large-follicle theca cells, aliquots (n = 4 pools) of freshly collected theca cells were treated with TRIzol Reagent (Life Technologies Inc., Gaithersburg, MD), RNA was collected, and real-time RT-PCR was used to quantify ALK5 mRNA that was normalized to constitutively expressed 18S ribosomal RNA (see below).

To determine the intrafollicular source of GDF9, aliquots (n = 6 pools) of freshly collected granulosa and theca cells were treated with TRIzol, RNA was collected, and real-time RT-PCR was used to quantify GDF9 mRNA that was normalized to constitutively expressed 18S ribosomal RNA (see below). Granulosa cells were prepared as oocyte-enriched or filtered using an embryo filter with a 75-µm-pore membrane (EZ-Way filter; A&E International, Alta Vista, KS). Granulosa cells retained on the filter were identified as "oocyte-enriched" granulosa cells, and those cells that passed through the membrane were identified as "filtered" granulosa cells.

Determination of Steroid Production and Cell Number

Medium was collected from individual wells and frozen at –20°C for subsequent determination of concentrations of progesterone and androstenedione in culture medium via RIA as previously described [32]. The intra- and interassay coefficients of variation were 12% and 18% for the progesterone RIA and 13% and 16% for the androstenedione RIA. Cells were gently washed twice with 0.9% saline (500 µl), exposed to 500 µl of trypsin solution (0.25% wt/vol; Sigma Chemical Co.) for 20 min at 25°C, and then scraped from each well. Cell aggregates were disrupted by pipetting the cell suspension back and forth through a 500-µl pipette tip three to five times, and cells were then diluted in 9 ml of 0.9% saline and counted using a Coulter counter (model Zm; Coulter Electronics, Hialeah, FL) as previously described [32, 33].

Immunofluorohistochemistry

Theca cells were cultured on 8-chamber slides as described for 24-well culture plates except that 1 x 105 cells were plated and 400 µl of medium used rather than 1 ml. After incubation for 2–4 days with various treatments, slides/cells were fixed in 10% formalin, rinsed three times with PBS, permeabilized using 0.01% Triton X-100 (Sigma Chemical Co.) in PBS for 30 sec, rinsed three times with PBS, and blocked for 30 min in PBS containing 0.2% normal rabbit serum (NRS; Linco Research, Inc., St. Louis, MO) and 1% BSA. Slides/cells were then incubated in the absence (negative control) or presence of a rabbit anti-porcine CYP17A1 polyclonal antiserum [45] at a 1:200 dilution (diluted in PBS containing 1% BSA, 0.1% NaN3, 0.2% NRS, and 2% normal bovine serum [Sigma Chemical Co.]) for 1 h at 25°C. After rinsing in PBS, cells were incubated sequentially with goat anti-rabbit immunoglobulins conjugated with Alexa Fluor 568 (Invitrogen Corp.) for 60 min at 25°C and then washed three times in PBS. Cells on slides were visualized using a confocal laser-scanning microscope (Leica Microsystems, Heidelberg, Germany). A positive reaction was demonstrated by a red fluorescence.

Real-Time RT-PCR Analyses

Theca and granulosa cells were lysed in 0.5 ml of TRIzol, RNA was extracted, and RNA quantity was determined spectro-photometrically at 260 nm as previously described [39, 46–48]. The target gene primer and probe sequences are listed in Table 1. Probes were synthesized over exon-exon junctions when possible. A BLAST search (http://www.ncbi.nlm.nih.gov/BLAST) was conducted to insure the specificity of the designed primers and probe. Furthermore, the RT-PCR product was run on an agarose gel to verify the length and size of the expected target gene, and the same RT-PCR cDNA sample was used to verify the amplified sequences.


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  		TABLE 1 Primers and probes used for mRNA quantification in RNA extracted from theca and granulosa cells of small and large follicles as described in Materials and Methods.

The differential expression of target gene mRNA in theca and granulosa cells was quantified using the one-step real-time RT-PCR reaction for the TaqMan Gold RT-PCR Kit (Applied Biosystems, Foster City, CA) as previously described [39, 46–48]. A ribosomal 18S RNA (18S rRNA) control kit (Applied Biosystems) was used as the internal control to normalize samples for the variation in amounts of RNA loaded [39, 46–48]. Based on preliminary optimization results, 50 or 100 ng of total RNA was amplified in a total reaction volume of 25 µl consisting of 200 nM forward primer, 200 nM reverse primer and 200 nM fluorescent (FAM/TAMRA) probe for each target gene, 10 nM of the 18S rRNA primers and 100 nM of the 18S rRNA VIC-labeled probe, along with 12.5 µl of TaqMan Master Mix without uracil N-gycosylase, and 1 U Multiscribe with RNase inhibitor (Applied Biosystems). Thermal cycling conditions were set to 30 min at 48.8°C for reverse transcription, 95°C for 10 min for AmpliTaq Gold Activations, 45 cycles at 95°C for 15 sec for denaturing, and 60°C for 1 min for annealing and extension (see Table 1 for Tm). All samples were run in duplicate. Relative quantification of target gene mRNA was expressed using the comparative threshold cycle method as previously described [39, 46–48]. Briefly, the {Delta}Ct was determined by subtracting the 18S Ct value from the target unknown value. For each target gene and within each experiment, the {Delta}{Delta}Ct was determined by subtracting the higher {Delta}Ct (the least expressed unknown) from all other {Delta}Ct values. Fold changes in target gene mRNA abundance were calculated as being equal to 2–{Delta}{Delta}Ct.

Transfection of Theca Cells

Theca cells (2 x 105 viable cells/well) were cultured in 24-well plates in medium supplemented with 10% FCS for 2 days as described earlier. After medium change, cells were incubated in 10% FCS medium without gentamycin for 24 h, wells were washed with serum-free medium, and cells were transfected with 250 ng of either CAGA or BRE reporter DNA per well using Lipofectamine 2000 in Opti-MEM-I (Invitrogen Corp.) as previously described [43, 44]. Briefly, to monitor transfection efficiency, the pCMV-β-galactosidase plasmid (50 ng) with reporter constructs was cotransfected for 4 h, wells were washed with serum-free medium, and cells were treated with GDF9 (300 ng/ml) or BMP4 (30 ng/ml) for 24 h in serum-free medium containing LH (30 ng/ml) and IGF1 (30 ng/ml). To harvest cells, lysis buffer (200 µl; Promega Corp.) was added into each well, and 30 µl of the supernatant was used for luciferase determination using a luminometer (Luminark microplate reader; Bio-Rad Laboratories, Inc.). To monitor transfection efficiency, 50 µl of the cell lysate were also used to measure the β-galactosidase activity. The reporter activity was expressed as the ratio of relative light unit:β-galactosidase activity.

Statistical Analyses

Each experiment contained three replicates per treatment, and each experiment was replicated three to four times with different pools of theca cells. Each pool of small-follicle theca cells was obtained from six to nine follicles collected from three to six cattle. Each pool of large-follicle theca cells was obtained from four to seven follicles collected from three to six cattle. Data are presented as the least squares means (± SEM) of measurements from nine to twelve culture wells. Main effects (i.e., hormone, dose, experimental replicate) and interactions were assessed using the general linear model procedure of SAS [49]. Steroid production was expressed as nanograms or picograms per 105 cells per 24 h, and cell numbers at the termination of the experiment were used for this calculation. If significant main effects were observed, specific differences among treatments in cell numbers, steroid production, promoter (luciferase) activity, and relative fold gene abundance were determined using the Fisher protected least significant difference procedure [50].

RESULTS

GDF9 Stimulates Proliferation of Theca Cells

Because IGF1 is a major stimulator of theca cell proliferation and steroidogenesis [32, 33, 51, 52], the effects of GDF9 on basal and IGF1-induced theca cell numbers were evaluated. In small-follicle theca cells, treatment with GDF9 caused a dose-dependent increase (P < 0.05) in basal (LH alone) and IGF1-induced cell numbers (Fig. 1A). All doses of GDF9 tested significantly increased theca cell numbers, and at the highest dose tested, GDF9 increased cell numbers by 2.5- to 3.2-fold (Fig. 1A). The stimulatory effect of GDF9 on theca cell numbers was demonstrable in the presence of LH and LH plus IGF1. In the absence of GDF9, IGF1 increased (P < 0.05) cell numbers by 46% compared with cells treated with only LH (Fig. 1A). In large-follicle theca cells, treatment with GDF9 (600 ng/ml dose only) weakly increased cell numbers (by 20%) in the presence of LH. In LH plus IGF1-treated theca cells from large follicles, a biphasic dose-response was evident with 150 ng/ml increasing (by 24%) cell numbers, whereas 300 and 600 ng/ml were without effect (Fig. 1B). In the absence of GDF9, IGF1 increased (P < 0.05) cell numbers by 24% compared with cells treated with only LH (Fig. 1B).


Figure 1
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  		FIG. 1 Stimulatory effects of GDF9 on theca cell proliferation. Theca cells were treated for 48 h with various doses of GDF9 in the presence of LH (30 ng/ml) without or with (+) IGF1 (30 ng/ml), and numbers of theca cells were determined by Coulter counting. A) Numbers of small-follicle theca cells. B) Numbers of large-follicle theca cells. Asterisks (**, *) indicate that mean (±SEM) differs from respective control (P < 0.05; n = 3 exp). C, Control.

Two-day treatment with LH alone, but not IGF1 alone, increased (P < 0.05) both progesterone and androstenedione production (Table 2). In contrast, IGF1 alone and in the presence of LH increased (P < 0.05) cell numbers, but LH alone had no effect (P > 0.10) on theca cell numbers (Table 2).


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  		TABLE 2 Effect of 2-day treatment of IGF1, LH, or both on the progesterone production, androstenedione production, and numbers of theca cells from large (≥8 mm) bovine follicles.

To confirm that increases in theca cell numbers induced by GDF9 were due to increased cell proliferation, changes in 3H-thymidine incorporation were monitored. In the presence (Fig. 2A) or absence (Fig. 2B) of LH (30 ng/ml), both IGF1 (30 ng/ml) and GDF9 (600 ng/ml) increased (P < 0.05) 3H-thymidine incorporation into small-follicle theca cells. Moreover, combined treatment with IGF1 and GDF9 further increased (P < 0.05) 3H-thymidine incorporation above that observed for either treatment alone (Fig. 2). The effect of IGF1 (i.e., 2- to 4-fold increases) on 3H-thymidine incorporation was greater (P < 0.05) than the effect of GDF9 (i.e., 1.7-fold increase) whether LH was present or absent in culture medium (Fig. 2).


Figure 2
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  		FIG. 2 Stimulatory effect of GDF9 on basal, LH-, and IGF1-induced theca cell proliferation/DNA synthesis. Small-follicle theca cells were serum starved and then treated for 40 h with 3H-thymidine and either no growth factor (Control), IGF1 (30 ng/ml; +IGF1), GDF9 (600 ng/ml; +GDF9), or GDF9 plus IGF1 (+GDF9+IGF1) in the presence (A; + LH) or absence (B; – LH ) of 30 ng/ml of LH. Cells were harvested, and 3H-thymidine incorporation into DNA was quantified. Values are means ± SEM of three separate experiments for each panel (n = 9). Within a panel, means without a common superscript differ (P < 0.05).

Purity of Theca Cells and GDF9 Effects on CYP17A1 Protein

To evaluate the purity of isolated theca cells and rule out fibroblast contamination, we used CYP17A1 and FSHR mRNA as markers for theca and granulosa cells, respectively. RNA from pools of theca and granulosa cells from small and large follicles were extracted and underwent real-time RT-PCR. Based on FSHR mRNA levels and CYP19A1 mRNA levels, small- and large-follicle theca cells averaged 5.6% and 8.3% contamination by granulosa cells, respectively (Table 3). Conversely, granulosa cells from small and large follicles averaged 1.08% and 0.08% contamination by theca cells, respectively, based on CYP17A1 mRNA levels (Table 3). Furthermore, immunofluorocytochemical staining of small-follicle theca cells revealed that over 95% of attached cells stained positive for CYP17A1 protein after 2-day exposure to 10% FCS (Fig. 3). Theca cells had greater (P < 0.05) LHR mRNA abundance than did granulosa cells, and large follicles had greater (P < 0.05) LHR mRNA abundance than small follicles in both theca and granulosa cells (Table 3).


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  		TABLE 3 Purity of theca cells isolated from small (3 to 6 mm) and large (≥8 mm) bovine follicles.*


Figure 3
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  		FIG. 3 CYP17A1 protein staining in small-follicle theca cells before and after GDF9 treatment. Small-follicle theca cells were cultured for 2 days in 10% FCS. Cells attached to wells were washed and then treated with 300 ng/ml of GDF9 in serum-free medium for an additional 2 days. Representative CYP17A1 immunofluorescent staining of theca cells after 1 or 2 days of culture in FCS (Day 0) dark field with fluorescence (A) and overlay on phase-contrast of cells (B); control NRS background staining revealed no specific fluorescence. C) Mean (±SEM) percentage of CYP17A1 immunofluorescent staining of theca cells after 0, 1, or 2 days in culture with and without GDF9.

To test if the stimulatory effect of GDF9 on cell proliferation involves alterations in the proportion of theca cells during culture, staining of CYP17A1 protein, a theca cell marker, was performed. Theca cells were obtained from small bovine follicles and cultured for 48 h in 10% FCS, followed by 1 or 2 days of control or GDF9 (600 ng/ml) treatment. Fluorescent staining for CYP17A1 protein was specific and stained over 85% of the theca cells (Fig. 3). As shown in Figure 3C, treatment with GDF9 for 1 or 2 days did not significantly alter the percentage of theca cells staining positive for CYP17A1 protein.

GDF9 Inhibits Steroidogenesis of Theca Cells

Androstenedione production. In the presence of LH, IGF1 increased (P < 0.01) androstenedione production (to 4.7-fold of controls) by theca cells from small follicles, and cotreatment with GDF9 caused a dose-dependent inhibition (P < 0.05) of this increase, with 600 ng/ml completely blocking the IGF1-induced increase (Fig. 4A). In the absence of IGF1, none of the doses of GDF9 affected (P > 0.10) LH-induced androstenedione production (Fig. 4A). In theca cells from large follicles, IGF1 increased androstenedione production 4-fold (Fig. 4B). GDF9 at 150 and 300 ng/ml had no effect, whereas 600 ng/ml of GDF9 weakly inhibited (by 17%; P < 0.05) the IGF1-induced increase in androstenedione production (Fig. 4B).


Figure 4
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  		FIG. 4 Inhibitory effects of GDF9 on theca cell androstenedione production. Theca cells were treated with various doses of GDF9 in the presence of LH (30 ng/ml) without or with IGF1 (30 ng/ml), and androstenedione concentrations in medium were measured by RIA. A) Androstenedione production by small-follicle theca cells. B) Androstenedione production by large-follicle theca cells. Within a panel, asterisks (**) indicate that mean (±SEM) differs from respective control (P < 0.05; n = 3 exp). C, Control.

Progesterone production. In the presence of LH, IGF1 increased (P < 0.001) progesterone production (to 6-fold of controls) by small-follicle theca cells, and GDF9 caused a dose-dependent inhibition (P < 0.05) of this increase, with 600 ng/ml completely blocking the IGF1-induced increase (Fig. 5A). In the absence of IGF1, only 600 ng/ml of GDF9 decreased (by 48%; P < 0.05) LH-induced progesterone production (Fig. 5A). In theca cells from large follicles, IGF1 increased progesterone production nearly 2-fold (Fig. 5B). GDF9 at 150 and 300 ng/ml had no effect, whereas 600 ng/ml of GDF9 weakly inhibited (P < 0.05) the IGF1-induced increase in androstenedione production (Fig. 5B).


Figure 5
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  		FIG. 5 Inhibitory effects of GDF9 on theca cell progesterone production. Theca cells were treated with various doses of GDF9 in the presence of LH (30 ng/ml) without or with IGF1 (30 ng/ml), and progesterone concentrations in medium were measured by RIA. A) Progesterone production by small-follicle theca cells. B) Progesterone production by large-follicle theca cells. Within a panel, asterisks (**, *) indicate that mean (±SEM) differs from respective control (P < 0.05; n = 3 exp). C, Control.

GDF9 Effects on Hormone Receptor and Steroid Enzyme mRNA in Small-Follicle Theca Cells

To test the cellular mechanisms underlying GDF9 suppression of androstenedione and progesterone biosynthesis, the ability of GDF9 to alter abundance of LHR, IGF1R, STAR, CYP11A1, and CYP17A1 mRNA was analyzed in cultured theca cells. Theca cells were obtained from small bovine follicles and cultured for 48 h in 10% FCS, followed by treatments with GDF9 and a related hormone BMP4. Treatment with GDF9 decreased (P < 0.05) the abundance of LHR mRNA by 25% (Fig. 6A), but had no effect (P > 0.10) on the abundance of IGF1R mRNA (Fig. 6B) in theca cells. In contrast, BMP4 increased (P < 0.05) abundance of LHR mRNA (by 40%; Fig. 6A), but had no effect on IGF1R mRNA (Fig. 6B). Treatment with LH plus IGF1 had no effect on the abundance of STAR mRNA (Control = 2.18 vs. LH + IGF1 = 1.99 ± 0.21). GDF9 did not influence the abundance of STAR mRNA (data not shown) and CYP17A1 mRNA (Fig. 7B), but decreased (P < 0.05) the abundance of CYP11A1 mRNA by 39% (Fig. 7A) in theca cells. In contrast, BMP4 had no effect on STAR mRNA (data not shown) but decreased (P < 0.05) abundance of both CYP11A1 mRNA (by 50%; Fig. 7A) and CYP17A1 mRNA (by 46%; Fig. 7B) in small-follicle theca cells.


Figure 6
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  		FIG. 6 Effect of GDF9 and BMP4 treatments on transcript levels for LHR (A) and IGF1R (B) mRNA in small-follicle theca cells. Small-follicle (3–6 mm) theca cells were treated for 24 h with serum-free medium alone (Control) or with GDF9 (300 ng/ml) or BMP4 (30 ng/ml) in the presence of LH (30 ng/ml) and IGF1 (30 ng/ml), and the various mRNAs were quantified by qRT-PCR as described in Materials and Methods. Within a panel, means (±SEM) without common letters differ (P < 0.05; n = 3 exp). Relative mRNA abundance was normalized to constitutively expressed 18S ribosomal RNA and expressed in arbitrary units.


Figure 7
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  		FIG. 7 Effect of GDF9 and BMP4 treatments on transcript levels for CYP11A1 (A) and CYP17A1 (B) mRNA in small-follicle theca cells. Small-follicle (3–6 mm) theca cells were treated for 24 h with serum-free medium alone (Control) or with GDF9 (300 ng/ml) or BMP4 (30 ng/ml) in the presence of LH (30 ng/ml) and IGF1 (30 ng/ml), and the various mRNAs were quantified by qRT-PCR as described in Materials and Methods. Within a panel, means (±SEM) without common letters differ (P < 0.05; n = 3 exp). Relative mRNA abundance was normalized to constitutively expressed 18S ribosomal RNA and expressed in arbitrary units.

Two-day treatment with LH plus IGF1 increased (P < 0.05) abundance of CYP11A1 mRNA in large-follicle theca cells (Table 4), but singular treatment of either LH or IGF1 did not significantly alter CYP11A1 mRNA abundance. In contrast, LH alone decreased (P < 0.05), whereas IGF1 alone had no effect (P > 0.30) on LHR mRNA abundance (Table 4). Concomitant treatment of LH and IGF1 attenuated the negative effect of LH on LHR mRNA abundance (Table 4). Abundance of IGF1R mRNA was not affected (P > 0.10) by LH, IGF1, or both (Table 4).


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  		TABLE 4 Effect of 2-day treatement of IGF1, LH, or both on the abundance of LHR, IGF1R, and CYP11A1 mRNAs in theca cells from large (≥8 mm) bovine follicles.*

GDF9 Effects on Promoter Activity in Small-Follicle Theca Cells

TGFB family ligands activate target cells through either SMAD2/3 or SMAD1/5/8 pathways [16, 17]. Because these two pathways can be characterized using CAGA and BRE promoter reporter constructs [18–20], we tested the ability of GDF9 to stimulate the CAGA versus BRE promoter in bovine theca cells. Theca cells were obtained from small bovine follicles and cultured for 48 h in 10% FCS, followed by transfection with the CAGA or BRE promoter and hormonal treatments for 24 h. As shown in Figure 8A, treatment with GDF9 (300 ng/ml) increased CAGA promoter activity 2.8-fold, but had no effect on BRE promoter activity. In contrast, treatment with BMP4 (30 ng/ml) increased BRE promoter activity 16-fold, but had no effect on CAGA promoter activity in transfected theca cells (Fig. 8B).


Figure 8
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  		FIG. 8 Effect of treatment with GDF9 and BMP4 on CAGA (A) and BRE promoter activities (B) in small-follicle theca cells. Small-follicle (3–6 mm) theca cells were transfected with either CAGA or BRE luciferase promoter and treated for 24 h with GDF9 (300 ng/ml) or BMP4 (30 ng/ml) in the presence of LH (30 ng/ml) and IGF1 (30 ng/ml) plus LH; cells were collected and luciferase and β-galactosidase assays (labeled β-Galac) conducted as described in Materials and Methods. Within a panel, means (±SEM) without common letters differ (P < 0.05; n = 3 exp). Relative promoter activity was normalized to β-galactosidase activity to correct for transfection efficiency.

ALK5 mRNA in Theca Cells from Small and Large Follicles

To determine if ALK5 mRNA abundance in small and large follicle theca cells differs, theca cells were isolated from both small and large follicles, and real-time QT-RT-PCR was conducted. As shown in Figure 9, theca cells from small follicles had greater (P < 0.05) abundance of ALK5 mRNA than large follicles.


Figure 9
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  		FIG. 9 Abundance of ALK5 mRNA in bovine theca cells. Theca cells were collected from small (3–6 mm) and large (8–22 mm) follicles, RNA was extracted, and real-time PCR was conducted. Values are means of four separate pools of cells and normalized to constitutively expressed 18S ribosomal RNA. *, mean (±SEM) differs (P < 0.05) from small-follicle theca cell mean.

GDF9 mRNA in Granulosa and Theca Cells

To determine if theca or granulosa cells contained GDF9 mRNA, cells were isolated from both small and large follicles, and real-time QT-RT-PCR was conducted. Theca cells from small and large follicles had no detectable GDF9 mRNA (data not shown), whereas filtered and oocyte-enriched granulosa cells from small follicles contained more GDF9 mRNA than those from large follicles (Fig. 10). Oocyte-enriched granulosa cells had significantly greater abundance of GDF9 mRNA than filtered granulosa cells (Fig. 10).


Figure 10
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  		FIG. 10 Abundance of GDF9 mRNA in granulosa cells. Granulosa cells collected from small (3–6 mm) and large (8–22 mm) follicles were separated on 70-µm filters to yield oocyte-enriched granulosa cells and granulosa cells without oocytes; RNA was extracted and real-time PCR conducted. Values are means of six separate pools of cells and normalized to constitutively expressed 18S ribosomal RNA. Means (±SEM) without common letters differ (P < 0.05).

DISCUSSION

The present studies revealed that: 1) GDF9 stimulates the proliferation of theca cells derived from small follicles in the absence and presence of IGF1, and these stimulatory effects of GDF9 were much less evident in large-follicle theca cells; 2) GDF9 inhibits small-follicle theca cell progesterone and androstenedione production induced by IGF1 and LH, and these inhibitory effects of GDF9 were much less evident in large-follicle theca cells; 3) the inhibitory effects of GDF9 treatment on androgen biosynthesis were associated with decreases in abundance of LHR and CYP11A1 mRNA, but GDF9 did not affect IGF1R, STAR, or CYP17A1 mRNA abundance in theca cells; 4) GDF9 likely transmits its intracellular signal via the SMAD2/3-dependent pathway and not the SMAD1/5/8 pathway; 5) ALK5 mRNA abundance was greater in theca cells from small rather than large follicles; and 6) granulosa cells but not theca cells may contain GDF9 mRNA, and levels of GDF9 mRNA appear to be greater in granulosa cells from small rather than large follicles. Thus, oocyte- and granulosa cell-derived GDF9 may play a role in increasing proliferation and suppressing differentiation of theca cells from small follicles.

A stimulatory effect of GDF9 was observed on basal and IGF1-induced theca cell proliferation, as measured by increased cell numbers and increased 3H-thymidine incorporation, and this stimulatory effect of GDF9 was not affected by LH. Although few studies have investigated theca cell proliferation using rodent models because of the difficulties involved in isolating pure theca cells, the present observations agree with a recent study showing treatment of GDF9 led to a small increase (by ~20%) in the number of human theca cells [31]. The minor GDF9 responses found by Yamamoto et al. [31] are likely due to the use of theca cells from large preovulatory follicles. The present findings that large-follicle theca cells were less responsive to the stimulatory effects of GDF9 suggest that GDF9 may preferentially stimulate theca cell proliferation during early follicle development. Consistent with a greater response in small-follicle theca, ALK5 (GDF9 receptor type I) mRNA abundance was greater in small-follicle rather than large-follicle theca cells. GDF9 increased numbers of theca cells and DNA synthesis in theca cells of small follicles, but did not alter the percentage of cells that expressed CYP17A1 protein or alter levels of CYP17A1 mRNA, indicating that GDF9 stimulates an increase in numbers of steroidogenic theca cells. Previous studies indicated that GDF9 signaling promotes CYP17A1 mRNA in murine ovaries, and thus GDF9 may stimulate the formation of theca-bearing follicles and/or the secretion of a theca cell recruitment factor [23, 53, 54]. The essential role of oocyte- and granulosa cell-derived GDF9 in supporting theca cell development in early follicles is underscored by findings in GDF9-null mice in which the theca layer fails to form and follicles do not develop beyond the primary stage [23]. Combined with studies showing that GDF9 treatment causes further development of primary follicles in hamsters [55], mice [26], and rats [27, 56], one can conclude that GDF9 stimulates early follicular growth, theca cell recruitment, and theca cell proliferation.

The present study indicates that the effects of GDF9 on follicular function may be dependent on the specific hormonal milieu. In particular, GDF9 had its greatest effect on decreasing theca cell progesterone and androstenedione production when both LH and IGF1 were present. Previously, 100–200 ng/ml of GDF9 decreased forskolin-induced (i.e., cAMP-induced) progesterone and androstenedione production by rat [30] and human [31] theca cells. GDF9 also inhibited LH-induced cAMP production (by 51%) in bovine theca cells (data not shown). In contrast, treatment with high doses (i.e., ≥250 ng/ml) of GDF9 increased basal progesterone and androstenedione production by cultured rat theca cells [30]. Abundance of STAR mRNA, the important cholesterol transport protein, was not influenced by GDF9 in the present study, but was inhibited by GDF9 in human theca cells [31]. Differences between the present and previous studies will require further elucidation, but are likely due to differences in culture conditions, species differences, and/or differences in the developmental stages of follicles from which theca cells are derived. Based on our mRNA studies, GDF9 appears to inhibit androgen production by inhibiting synthesis of LHR and CYP11A1 mRNA rather than CYP17A1 mRNA. In the theca cell model used in the present study, LH alone (but not IGF1) stimulates steroidogenesis, but LH is needed for IGF1 to stimulate steroidogenesis. This stimulatory effect of LH is via induction of CYP11A1 mRNA and not IGF1R mRNA. Thus, GDF9 reduces LH-induced progestin precursor formation and subsequently reduces androgen formation by CYP17A1.

The TGFB family ligands initiate signaling by assembling type I and type II serine/threonine kinase receptor complexes that activate two main SMAD pathways [6, 16, 17, 57]. Studies using granulosa cells indicated that GDF9 interacts with BMPR2 (type II receptors), followed by the specific activation of the type I receptor, ALK5, and subsequent phosphorylation of the downstream SMAD2/3 proteins characterized by the stimulation of the CAGA promoter [6, 43, 58]. In comparison, BMP4 and BMP2 bind to BMPR2 and the type I receptors, ALK3 and ALK6, leading to the activation of the SMAD1/5/8 pathway [16, 59] and subsequent activation of the BRE promoter [43, 58, 60]. Our studies using transfected bovine theca cells indicate that GDF9 stimulates the activities mediated by the CAGA, but not the BRE promoter, and are consistent with previous studies using cultured granulosa cells of bovine, rodent, and human origin [43, 44, 58, 60]. For the first time, abundance of ALK5 mRNA in theca cells was demonstrated to be greater in small follicles than large follicles, and this may explain, in part, why small-follicle theca cells responded better to GDF9 than large-follicle theca cells, and indicates that the GDF9 response system (i.e., ALK5) may be regulated during follicle growth. In comparison, a combination of estradiol and FSH increased abundance of ALK5 and BMPR2 mRNA in bovine granulosa cells [57]. Whether LH and/or IGF1 alters ALK5 or BMPR2 mRNA abundance in theca or granulosa cells will require further elucidation. Furthermore, whether the doses of GDF9 used in the present and previous studies are similar to levels present within the follicles remain to be determined. Because granulosa cells may be capable of synthesizing GDF9 in pigs [10], humans [31], and cattle (present study), developing follicles in those species may not have to rely on the oocyte as its sole source of GDF9. Caution should be made when interpreting the GDF9 mRNA data of the present study, as broken oocytes may have contaminated the "pure" fraction of granulosa cells.

In conclusion, the present studies demonstrated a follicle developmental stage-dependent effect of GDF9 on theca cell function. Granulosa cells, but not theca cells, contained GDF9 mRNA, and levels of GDF9 mRNA appear to be greater in granulosa cells of small rather than large follicles. Theca cells from small follicles were more responsive to both the stimulatory effect of GDF9 on cell proliferation and the inhibitory effect of GDF9 on thecal steroidogenesis and contained a greater abundance of ALK5 mRNA than theca cells from large follicles. The decreased responses and abundance of ALK5 mRNA in theca cells from small to large follicles suggests that the GDF9 response system in theca cells may be regulated during follicle growth. The highly pure theca cell model used in the present study should prove valuable for further study of the molecular mechanism underlying GDF9 actions in theca cells and the roles of other paracrine factors that may act on the theca interna.

ACKNOWLEDGMENTS

The authors thank A. Grado, M. Aleman, and C. Klein for technical assistance; Creekstone Farms (Arkansas City, KS) for their generous donations of bovine ovaries; Dr. Robert Matts for use of the luminometer; and Dr. Charlotte Ownby of the OSU Microscopy Laboratory for assistance with the confocal laserscanning microscopy and capture of fluorescent images.

FOOTNOTES

1Approved for publication by the Director, Oklahoma Agricultural Experiment Station, and supported in part under project H-2510. This project was also supported by the National Research Initiative Competitive Grant 2005-35203-15334 from the USDA Cooperative State Research, Education, and Extension Service, the National Institute of Child Health and Human Development, and the National Institutes of Health through Cooperative Agreement U54 HD31398 as part of the Specialized Cooperative Centers Program in Reproductive Research. Back

Correspondence: 2Correspondence: FAX: 405 744 7390; e-mail: igf1leo{at}okstate.edu

Received: 14 June 2007.

First decision: 16 July 2007.

Accepted: 17 October 2007.

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F. Paradis, S. Novak, G. K Murdoch, M. K Dyck, W. T Dixon, and G. R Foxcroft
Temporal regulation of BMP2, BMP6, BMP15, GDF9, BMPR1A, BMPR1B, BMPR2 and TGFBR1 mRNA expression in the oocyte, granulosa and theca cells of developing preovulatory follicles in the pig
Reproduction, July 1, 2009; 138(1): 115 - 129.
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J. L Juengel, N. L Hudson, M. Berg, K. Hamel, P. Smith, S. B Lawrence, L. Whiting, and K. P McNatty
Effects of active immunization against growth differentiation factor 9 and/or bone morphogenetic protein 15 on ovarian function in cattle
Reproduction, July 1, 2009; 138(1): 107 - 114.
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 EndocrinologyHome page
M. Orisaka, J.-Y. Jiang, S. Orisaka, F. Kotsuji, and B. K. Tsang
Growth Differentiation Factor 9 Promotes Rat Preantral Follicle Growth by Up-Regulating Follicular Androgen Biosynthesis
Endocrinology, June 1, 2009; 150(6): 2740 - 2748.
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